Edited by Henry C. Harpending, University of Utah, Salt Lake City, UT, and approved March 27, 2003 (received for review January 20, 2003)

Abstract
During the late Pleistocene, early anatomically modern humans coexisted in Europe with the anatomically archaic Neandertals for some thousand years. Under the recent variants of the multiregional model of human evolution, modern and archaic forms were different but related populations within a single evolving species, and both have contributed to the gene pool of current humans. Conversely, the Out-of-Africa model considers the transition between Neandertals and anatomically modern humans as the result of a demographic replacement, and hence it predicts a genetic discontinuity between them. Following the most stringent current standards for validation of ancient DNA sequences, we typed the mtDNA hypervariable region I of two anatomically modern Homo sapiens sapiens individuals of the Cro-Magnon type dated at about 23 and 25 thousand years ago. Here we show that the mtDNAs of these individuals fall well within the range of variation of today's humans, but differ sharply from the available sequences of the chronologically closer Neandertals. This discontinuity is difficult to reconcile with the hypothesis that both Neandertals and early anatomically modern humans contributed to the current European gene pool.

The origin of anatomically modern humans (a.m.h.) is traditionally explained by two contrasting models. The multiregional model, in its original formulation (1, 2), proposed that modern humans evolved in the last two million years as a single polytypic species, through the independent appearance of modern traits in different areas at different times. Recent adjustments of this model (3–5) retained the concept of a single evolving species, but argue that modern forms expanding from Africa may have mixed, even extensively, with archaic forms, such as Neandertals in Europe. Variants of the multiregional model differ in the extent of predicted admixture between anatomically modern and archaic humans, but all of them assume a dual ancestry, archaic and modern, in the current human gene pool, and therefore some continuity over time between these forms.

The Out-of-Africa model (6), on the other hand, suggests that a.m.h. first arose in Africa some 150,000 years ago and then dispersed replacing archaic forms (Neandertals in Europe). This model does not imply any hypothesis regarding the structure of archaic humans populations, nor that modern humans migrating from Africa were a biologically distinct species. It only assumes that modern traits evolved recently in a single region, Africa, and the vast majority of the genomes of current human populations can be traced back to these African migrants. This hypothesis does not seem to differ radically from Relethford's (4) view of multiregional evolution, whereby the Neandertal's contribution to the modern European gene pool may have been small, yet nonzero.

Analyses of morphological traits, Neandertal ancient DNA and modern DNA (e.g., refs. 7–9), appear to support a recent African origin of all humankind. However, it has been argued that patterns of genetic diversity are not incompatible with a multiregional model (5, 10–12). For instance, Nordborg (10) showed that the differences between Neandertal's and modern mitochondrial sequences are sufficient to rule out random mating between them, but not more complicated models of interbreeding. Those results reflect the existing uncertainties on the European demographic history of the last 30,000 years. Clearly, genetic typing of the earliest a.m.h. in Europe, sometimes referred to as Cro-Magnons or Cro-Magnoid from the site in France where they were first discovered, is a crucial step for solving this question (13–15), because that would allow a genetic comparison between individuals who lived at a much shorter (ideally, zero) time distance. The Out-of Africa model, in fact, predicts genetic discontinuity between Neandertals and early a.m.h. (the former being a separate lineage replaced by the latter) and genetic continuity along the a.m.h. lineages from the Upper Palaeolithic until the present. The multiregional models, on the contrary, predict at least some level of genetic continuity from the archaic Neandertal forms to the almost contemporary Cro-Magnon forms up to today's Europeans.

In this study, we typed the hypervariable region I (HVRI) of the mitochondrial genome (360 bp) from the bones of two early a.m.h. of the Cro-Magnon type from Southern Italy. We validated the sequences obtained through a number of biochemical tests, and we compared them with those of four Neandertal (8, 16–18) specimens and with a large data set of modern human sequences (19, 20).